Klaus Steinmüller

The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts

The proton‐pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for the synthesis of ATP. Closely related forms of this complex exist in the mitochondria of eucaryotes and in the plasma membranes of purple bacteria. The minimal structural framework common to the mitochondrial and the bacterial complex is composed of 14 polypeptides with 1 FMN and 6–8 iron‐sulfur clusters as prosthetic groups. The mitochondrial complex contains many accessory subunits for which no homologous counterparts exist in the bacterial complex. Genes for 11 of the 14 minimal subunits are also found in the plastidial DNA of plants and in the genome of cyanobacteria. However, genes encoding the 3 subunits of the NADH dehydrogenase part of complex I are apparently missing in these species. The possibility is discussed that chloroplasts and cyanobacteria contain a complex I equipped with a different electron input device. This complex may work as a NAD(P)H: or a ferrodoxin:plastoquinone oxidoreductase participating in cyclic electron transport during photosynthesis.

The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis

Abstract. The activity of the bidirectional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was found not to be regulated in parallel to respiration but to photosynthesis. A mutant with a deletion in the large hydrogenase subunit gene (hoxH), which contains the active site, was impaired in the oxidation of photosystem I (PSI) when illuminated with light, which excites either PSI alone or both photosystems. The fluorescence of photosystem II (PSII) of this mutant was higher than that of wild-type cells. The transcript level of the photosynthetic genes psbA, psaA and petB was found to be different in the hydrogenase-free mutant cells compared to wild-type cells, which indicates that the hydrogenase has an effect on the regulation of these genes. Collectively, these results suggest that the bidirectional hydrogenase functions as a valve for low-potential electrons generated during the light reaction of photosynthesis, thus preventing a slowing down of electron transport. This conclusion is supported by growth curves demonstrating that the mutant cells need more time to adapt to changing light intensities. Investigations of the wild-type and ΔhoxH strains strongly suggest that Synechocystis contains only the bidirectional hydrogenase, which seems to be essentially insensitive to oxygen.

Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation

Abstract Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1–5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.

Differential expression of plastome-encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C4 plant Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone oxidoreductase is involved in cyclic electron transport

Cyanobacteria and plastids harbor a putative NAD(P)H- or ferredoxin-plastoquinone oxidoreductase that is homologous to the NADH-ubiquinone oxidoreductase (complex I) of mitochondria and eubacteria. The enzyme is a multimeric protein complex that consists of at least 11 subunits (NDH-A-K) and is localized in the stroma lamellae of the thylakoid membrane system. We investigated the expression of the different subunits of the enzyme in mesophyll and bundle-sheath chloroplasts of Sorghum bicolor [L.] Moench, a C4 plant of the NADP-malic enzyme type. The relative amounts of the subunits NDH-H, -J and -K were strongly increased in bundle-sheath plastids as compared to mesophyll plastids. This increase was accompanied by enhanced transcript levels for all subunits except NDH-I. Because the main function of the protein complexes in the thylakoid membranes of bundle-sheath chloroplasts (photosystem I, cytochrome b6/f-complex and ATPase) is the generation of ATP for CO2 fixation via cyclic electron transport, we conclude that the NAD(P)H/ferredoxin-plastoquinone oxidoreductase is an essential component of the cyclic electron-transport pathway in chloroplasts.

Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts

The plastid genomes of higher plants contain eleven reading frames (ndhA-K) that are homologous to genes encoding subunits of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I). The carboxyterminal end of the NDH-H subunit from rice (Oryza sativa L.) was expressed as a fusion protein in Escherichia coli and antibodies against the fusion protein were generated in rabbits. The antibody was used to study the expression of NDH-H, and the following results were obtained: (i) NDH-H is expressed in mono- and dicotyledonous plants, (ii) NDH-H is localized on the stroma lamellae of the thylakoid membrane and (iii) NDH-H is expressed in etioplasts. Together with the finding that two other ndh genes (ndhI and ndhK) are expressed in plastids, these results point to the existence of an NAD(P)H-plastoquinone-oxidoreductase on the thylakoid membrane. The possible function of the enzyme in plastids is discussed and it is suggested that it works in balancing the ATP/ADP and the NADPH/NADP ratios during changing external (i.e. light) or internal (i.e. ATP and NADPH demands of biosynthetic pathways of the plastid) conditions.

Cyanobacteria contain a mitochrondrial complex I-homologous NADH-dehydrogenase

Thylakoid and cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803 were purified by sucrose gradient centrifugation. Both membranes oxidize NADH in a rotenone‐sensitive reaction. Antibodies prepared against psbG/ndhK and ndhJ fusion proteins detect the corresponding polypeptides in both membrane preparations. This demonstrates that a NADH‐dehydrogenase, homologous to the mitochondrial NADH‐ubiquinone‐oxidoreductase (complex I of the respiratory chain) is present in cyanobacteria. The NADH‐dehydrogenase can be solubilized with the detergent β‐D‐dodecylmaltoside. Sedimentation analysis of the solubilized enzyme on a sucrose gradient indicates that it is a multisubunit protein complex.

Immunopurification of a subcomplex of the NAD(P)H-plastoquinone­ oxidoreductase from the cyanobacterium Synechocystis sp. PCC6803

An antibody against the NDH‐K subunit of the NAD(P)H‐dehydrogenase from the cyanobacterium Synechocystis sp. PCC6803 was used to isolate a subcomplex of the enzyme from Triton X‐100 solubilized total membranes by immunoaffinity chromatography. The isolated subcomplex consisted of seven major polypeptides with molecular masses of 43, 27, 24, 21, 18, 14 and 7 kDa. The amino‐terminal amino acid sequences of the polypeptides were determined. By comparing the sequences with the amino acid sequences deduced from DNA, three proteins were identified as NDH‐H (43 kDa), NDH‐K (27 kDa) and NDH‐I (24 kDa). A fourth subunit (NDH‐J, 21 kDa) was identified by Western blot analysis with an NDH‐J antibody.

Differential transcription of plastome-encoded genes in the mesophyll and bundle-sheath chloroplasts of the monocotyledonous NADP-malic enzyme-type C4 plants maize and Sorghum

The transcription of plastome-encoded genes in mesophyll and bundle-sheath chloroplasts of the monocotyledonous NADP-malic enzyme-type C4 species Zea mays L. (maize) and Sorghum bicolor (L.) Moench. was investigated. RNA accumulation and transcription were assayed starting from isolated mesophyll and bundle-sheath chloroplasts and using quantitative northern and run-on transcription analysis. Determination of the mesophyll to bundle-sheath ratios of transcript abundance in maize and Sorghum chloroplasts showed that the mRNAs of the plastome-encoded photosystem II genes analysed (psbA, psbB, psbD, psbH and psbE/F) varied from 2.5- to 4.0-fold (maize) and 3.1- to 5.2-fold (Sorghum), respectively. The rbcL transcript, in contrast, was more abundant in bundle-sheath chloroplasts of both species, about 3-fold in maize and more than 10-fold in Sorghum. On the other hand, transcripts of genes encoding the 16S ribosomal RNA (r16) and subunits of photosystem I (psaA) and the cytochrome b/f complex (petB, petA) accumulated to similar levels in both types of chloroplasts. Determination of absolute transcript levels for rbcL and psbA in chloroplasts from maize and Sorghum demonstrated that for both genes, the mesophyll to bundle-sheath differences in transcript abundance were more pronounced in Sorghum. Measurements of the transcriptional activities of rbcL and psbA showed that the transcription rate of rbcL is higher in bundle-sheath chloroplasts while psbA is more actively transcribed in mesophyll chloroplasts. The differences in the transcription rates between the two chloroplast types were again more pronounced in Sorghum, thus reflecting the differences between maize and Sorghum in the relative levels of the rbcL and psbA transcripts. However, although transcription rate and mRNA abundance are correlated, they did not exactly match one another. This indicates additional regulation of transcript abundance at the level of RNA stability.

PEG-mediated plastid transformation in higher plants

SummaryPlastids are surrounded by an envelope consisting of a double membrane. This barrier has to be penetrated or overcome by the DNA when transforming the plastome. Both the biolistic and polyethylene glycol-mediated transformation techniques accomplish this task, albeit by different mechanisms. We were the first laboratory to successfully use the polyethylene glycol (PEG)-method for plastid transformation, yet we use the particle gun when appropriate. In this report we compare the two methods and discuss their shortcomings and advantages. Plastid transformations with various constructs, mainly using theaadA gene as a selective marker, were performed. We point to potential problems likely to be encountered during the transformation and selection processes and offer possibilities for improvement. We give further examples of the successful application of plastome transformation and show its merits in addressing biological questions concerning the elucidation of plastid sequences of unknown function and the control of plastid gene expression.

Cloning and transcription analysis of the ndh(A-I-G-E)gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803.

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA,-I, G,-E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40–68% identity) than to the corresponding mitochondrial subunits (17–39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.